Journal of Medical and Biological Engineering, 34(1): 44-48
44
Improvement of Osteoblast Adhesion Through Polarization of
Plasma-Sprayed Hydroxyapatite Coatings on Metal
Miho Nakamura1,*
Akira Kobayashi2
Naohiro Horiuchi1
Akiko Nagai1,3
Kosuke Nozaki1,3
Kimihiro Yamashita1
1
Department of Inorganic Materials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 113, Japan
Development Base of Advanced Materials Development and Integration of Novel Structured Metallic and Inorganic Materials, Joining and Welding
Research Institute (JWRI), Osaka University, Osaka 565, Japan
3
Department of Material Biofunctions, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 113, Japan
2
Received 1 Feb 2013; Accepted 13 Jun 2013; doi: 10.5405/jmbe.1450
Abstract
Hydroxyapatite coatings (HACs) have long been applied to orthopedic and dental implants made of titanium and
its alloys because of their high biocompatibility and osteoconductivity. We have recently demonstrated that the charged
surfaces on HAC induced by polarization enhance mineral deposition in simulated body fluid and osteoconductive
capabilities in vivo. The present study evaluates the effects of the electrical polarization of HAC on surface
characteristics and osteoblast adhesion. It was found that electrical polarization has no effect on surface roughness and
crystallinity. Morphological observations and quantitative analyses of adhered osteoblasts on HACs revealed that the
cell areas positively stained for actin, which indicates the degree of cell spreading, were distinctly larger on negatively
and positively charged HAC than that on uncharged HAC.
Keywords: Hydroxyapatite coating, Polarization, Osteoblast adhesion
1. Introduction
In clinical applications, hydroxyapatite (HA) is coated
onto metal substrates, such as titanium (Ti), Ti alloys, and
stainless steel (SUS 316), to offset its mechanical weakness.
Various methods of fabricating HA coatings (HACs) have been
developed for dental and orthopedic implants, such as plasma
spraying, radio-frequency (RF) magnetron sputtering, dip
coating, electrochemical deposition, pulsed-laser deposition,
and electrophoresis deposition methods. HACs have long been
applied to orthopedic and dental implants made of metals
because of their high biocompatibility and osteoconductivity
that is the formation of new bone in the vicinity of the
implanted biomaterials. The osteoconductivity of HACs has
been found to be inferior to those of autografts and thus HAC is
unsuitable for filling the wide clearance between implants and
bone tissues with newly formed bone due to a slow
osteoconduction process. Osteoconduction is considered to
progress in six stages at the interface between the implanted
biomaterials and injured hard tissues: (1) serum adsorption; (2)
recruitment of various cell types; (3) attachment, motility, and
* Corresponding author: Miho Nakamura
Tel: +81-3-52808015
E-mail: [email protected]
proliferation of various cell types; (4) osteoblast differentiation
and osteoid production; (5) matrix calcification; and (6) bone
remodeling [1,2]. The adhesion of osteoblasts to the
biomaterials is crucial in the regulation of the subsequent
differentiation and the formation of the extracellular matrix
following spreading and motility [3]. The present study thus
focuses on the analysis of osteoblast behavior near
biomaterials.
The mechanism of cell adhesion on biomaterials varies
according to the type of substrate. Human osteoblast-like cells
initially attach and spread more quickly on HA than on
titanium [3]. HA and titanium surfaces, furthermore, influence
gene expression at an early phase of adhesion as well as at the
later phases of proliferation and differentiation [4]. These
behaviors of osteoblasts can be attributed to the differences in
surface characteristics, which affect the signal transduction
pathways. The signal transduction pathways involved in the
adhesion of osteoblasts on HA and titanium were confirmed by
the subsequent expression of v1 integrins [3].
The adhesion of osteoblasts primarily depends on the
surface characteristics of the biomaterials involved and
stimulation from outside the cells. The surface characteristics
are affected by surface roughness, surface crystallinity [4-6],
constituent elements at the surface, and the incorporation of
ions such as carbonate or fluorine [7,8]. Electron-induced
surface energy modifications such as surface photovoltage
J. Med. Biol. Eng., Vol. 34 No. 1 2014
spectroscopy can be used to improve surface characteristics [9].
Stimulation from outside the cells includes electrical
stimulation such as capacitive coupling, inductive coupling,
and combined electromagnetic fields, which affect osteoblast
attachment, adhesion, and motility [9]. Cell shapes on
substrates depend on the integrin-mediated cytoskeletal and
signal transduction molecules, such as actin filaments and
vinculin [10,11], and are important during cell-substrate
adhesion for subsequent cell behaviors such as proliferation
and differentiation [8,12]. Therefore, the present study focuses
on adhered cell shapes, as indicated by the actin structure, to
study osteoblast adhesion.
We have recently demonstrated that the charged surfaces
on dense HA induced by polarization [13-17] enhance
osteoconductive capabilities in vivo [18,19] and that the
charged surfaces on HAC induced by polarization enhance
mineral deposition in simulated body fluid (SBF) [20] and
osteoconductive capabilities in vivo [21]. Additionally,
polarized HA affects both hard and soft tissues. It enhanced the
blood vessel regeneration of a vascularly injured model [22]
and epidermal recovery from full-thickness skin wounds in vivo
[23]. Polarization treatment is thus considered to affect cell
behaviors. The initial adhesion and motility of osteoblast-like
cells on polarized HA were accelerated in vitro [24]. Although
the polarization treatment enhanced new bone formation in the
vicinity of the polarized HAC in vivo, the mechanisms of the
effects induced by the polarization treatment on osteoblast
behavior were not completely identified.
In a previous study, -tricalcium phosphate (-TCP) as a
starting material was coated onto Ti by the plasma-spraying
method and transformed into the HA phase through
hydrothermal treatment [20]. The different coating method was
tried to intend to a wide range of applications. The HA was
directly coated onto SUS 316 using the gas-tunnel-type
plasma-spraying method. The electrical properties of HA
ceramics were highly sensitive to the crystal structures and
microstructures. In particular, the electrical polarization of HA
was appreciably influenced by the crystal structure in the
vicinity of protons and the grain boundary in the
microstructure. The present study characterizes HAC with or
without thermal treatment and the effects of electrical
polarization of HAC on osteoblast adhesion.
45
and heated HAC (H-HAC) specimens were observed by
scanning electron microscopy (SEM; Hitachi S-2400, Japan).
HAC and H-HAC specimens were characterized by X-ray
diffraction (XRD). XRD measurements were performed for
phase analysis at room temperature (RT) with CuK radiation at
40 kV and 40 mA on a diffraction spectrometer (Philips
PW1700,
Netherlands)
equipped
with
a
graphite
monochromator.
The HA-coated SUS 316 blocks were electrically polarized
with a pair of platinum electrodes at 300 C in a DC electric
field of 30 V for 1 h in air according to our previous work
(Fig. 1) [16,20]. The temperature for electrical polarization was
decided to provide the appearance of the maximum point in
thermally stimulated depolarization current (TSDC) spectrum
less than 600 C. The electrically polarized HAC are either
negatively charged (N-HAC) or positively charged (P-HAC).
The surface of HAC heated at 300 C was denoted as H-HAC.
Figure 1. Schematic illustration of experimental procedure. HAC
specimens were electrically polarized in a DC field of 30 V
with a pair of platinum electrodes in air at 300 C for 1 h. The
electrically polarized HAC was either negatively (N-HAC) or
positively charged (P-HAC). The surface of HAC heated at
300 C was denoted as H-HAC.
Polarization of the HA specimens was verified by TSDC
measurement. The TSDC measurements were carried out
according to our previous study [20,21] in air from RT to
600 C at a heating rate of 5.0 C/min. The depolarization
current was measured with a Hewlett-Packard 4140B pA meter.
The values of the polarization charge (Qp) were calculated from
the TSDC spectra using:
Qp =1/  J (T) dT
(1)
2. Materials and methods
where J (T) is the measured dissipation current density at
temperature T and  is the heating rate.
2.1 Sample preparation
2.2 Osteoblast culture
HA powder as starting powder for the plasma spraying
was synthesized from the analytical-grade reagents calcium
hydroxide and phosphoric acid by the wet method [16]. The
metal substrate was stainless steel (SUS-316) blocks
(1 cm  1 cm  3 mm). The HAC of the metal substrates were
prepared using the gas-tunnel-type plasma-spraying method
developed by Arata et al. [25]. The thickness of the HAC layers
was ca. 30 m.
Surface characterization was performed to investigate the
differences before and after heat treatment. Surfaces of HAC
Osteoblast cells (MC3T3-E1 cell line) obtained from the
RIKEN Cell Bank (Tsukuba, Japan) were used for the
osteoblast adhesion assay. This cell line is widely used in the
biomaterials field. It was easy to observe the morphology of
each cell adhered on the HAc specimens. The cells were
maintained in -modified minimum essential medium (MEM), supplemented with 10% fetal bovine serum (FBS),
50 units/ml penicillin, and 50 g/ml streptomycin in a
humidified atmosphere of 5% CO2 in air at 37 C. After
46
Osteoblast Adhesion on Polarized Hydroxyapatite Coating
reaching 70% confluency, the cells were detached by treatment
with 0.25% trypsin and then seeded into culture plates at a
density of 0.5 × 104 cells/cm2 in -MEM containing 10% FBS,
50 units/ml penicillin, and 50 g/ml streptomycin. The medium
was changed every 3-4 days.
2.3 Osteoblast adhesion assay
The HAC specimens were sterilized with 70% ethanol and
immersed in the cell culture medium for 30 min. The cells were
seeded into HAC specimens at a density of 0.5 × 104 cells/ml in
-MEM containing 10% FBS, 50 units/ml penicillin, and
50 g/ml streptomycin. After 1 h and 3 h, the cells on the HAC
samples were fixed with 4% paraformaldehyde and
permeabilized with 0.1% Triton X-100 in phosphate-buffered
saline (PBS). The cells were stained with rhodamine phalloidin
and Hoechst. Fluorescent signals were observed using a
fluorescence microscope (Olympus IX71, Japan). The cell
areas positively stained for actin were measured using
MetaMorph software. The measurement was performed for a
minimum of 50 cells on each surface.
Accurate quantification in three HAC samples was
achieved using three independent experiments. The differences
were analyzed using one-way or two-way analysis of variance
(ANOVA). Student’s t-test (paired or unpaired) was used to
ascertain the differences between the two groups. The statistical
significance was defined as p < 0.05.
3. Results and discussion
(a)
(b)
Figure 3. SEM images of the surfaces of (a) HAC and (b) HAC heated
at 300 C. Scale bar = 100 m.
As shown in Fig. 4, the TSDC curve of electrically
polarized H-HAC increased at ca. 400 C, reached a shoulder
point at ca. 600 C, and then gradually increased. The shoulder
of the TSDC curve indicates depolarized current being released
from the electrically polarized H-HAC specimen. The stored
charges were calculated from the TSDC spectra at 52 C·cm-1.
The maximum current density and the stored charges calculated
from TSDC spectra of the HAC were higher than those of
dense HA [16]. The value of the stored charges of HAC was
approximately 3.5 times higher than that for dense HA. It was
previously suggested that the polarization and depolarization
can be attributed to the migration of the protons of OH- in
apatite columnar channels [16]. According to a study on the
complex impedance of HA [16], protons migrate through both
the grains and grain boundary. The resistances of the grain
boundaries were higher than those of the grains, indicating that
the grain boundaries were obstacles to proton transfer.
The XRD patterns of HAC and H-HAC were highly
consistent with the published data of HA (ICDD No. 9-432),
indicating that the HAC surfaces consisted of a single phase of
hexagonal HA before and after heating at 300 C (Fig. 2). SEM
images of the surfaces of HAC and H-HAC are shown in Fig, 3.
The surfaces were partially covered with spherical crystals
approximately 30 m in diameter and molten grains under the
spherical crystals were observed. The surface characteristics of
H-HAC revealed that heating at 300 C had no effect on
surface crystallinity, crystal phase, and morphology.
Figure 4. Elecrical polarizability estimated from TSDC measurements.
TSDC spectra of HAC electrically polarized at 300 C were
obtained in a DC field of 30 V for 1 h.
Figure 2. XRD patterns of (a) HAC and (b) HAC heated at 300 C.
Because the number of protons was proportional to
volume, HAC should have a much smaller number than that of
HA. However, the value of the stored charges of HAC was
much higher than that of HA. It was suggested that the defects
of calcium ions stimulate proton migration and act as possible
trap sites of protons [13]. Moreover, the OH- ions in HAC were
unstable due to the heat history during plasma spraying, such as
rapid cooling after heating at a high temperature. Consequently,
the protons easily migrated.
J. Med. Biol. Eng., Vol. 34. No. 1 2014
The longer migration distance of the protons is considered
to be another reason for the high stored charges of HAC. It was
reported that the grain boundaries are obstacles for proton
migration during the polarization of dense HA [13]. The
observed average grain size of HA was approximately 1 m
[20]. Some HA grains had obvious boundaries and the average
grain sizes were approximately 30 m in diameter (Fig. 3).
Most of the HA grains were molten because of the hightemperature plasma flame, and the grain boundary was
indistinct. Therefore, the higher value of the stored charges of
HAC can be attributed to the complicated structures of HAC.
Figure 5 shows actin and nuclei labeling of the cells after
seeding onto the HAC specimens for 1 h and 3 h. During
cultivation 1 h after cell seeding, the cells that adhered on HHAC showed a round or spherical configuration. The cells that
adhered on N-HAC and P-HAc showed a spindle- or fan-like
shape after 1 h of cultivation. During subsequent incubation
after 3 h of cell seeding, the cells that adhered on H-HAC were
spread out and showed a slightly spindle-like or rectangular
shape. An accumulation of actin filaments in the periphery of
lozenge-shaped cells was observed for both N-HAC and PHAC. In addition, bundles of actin fibers forming stress fibers
appeared in the cells on N-HAC and P-HAC as the attached
cells spread. Well defined stress fibers showing a regular
arrangement with particular polarities were found in some cells
grown on N-HAC and P-HAC. In some cells on N-HA and
P-HA, the actin filaments were mostly distributed near the edge
of pseudopodia-like structures and formed weak bundles of
stress fibers.
47
cell area were observed between N-HAC and P-HAC. It would
be useful to further investigate whether the polarization affects
the proliferation and differentiation of osteoblasts, which are
subsequent events in the osteoconduction process.
Figure 6. Cell areas positively stained for actin for the three HAC
specimens. The cell area was significantly larger on N-HAC
and P-HAC compared to that on HAC at 1 h (*p < 0.005
compared with HAC) and 3 h (*p < 0.001 compared with
HAC) after seeding.
4. Conclusion
The surface characteristics of HAC specimens reveal that
electrical polarization has no effect on surface roughness and
crystallinity. Morphological observations and quantitative
analyses of adhered osteoblasts on HAC specimens revealed
that the cell areas positively stained for actin, which indicates
the degree of cell spreading, were distinctly larger on N-HAC
and P-HAC than that on H-HAC.
Acknowledgments
This work was partly supported by the Project of
Advanced Materials Development and Integration of Novel
Structual Metallic and Inorganic Materials, the Shiseido Female
Researcher Science Grant, the Asahi Glass Foundation, and
Grants-in-Aid from the Japan Society for the Promotion of
Science (#23300178).
Figure 5. Morphology of adhered osteoblasts on HAC, N-HAC, and PHAC. Fluorescence images with actin and nuclei staining of
the cells cultured on the HAC specimens for 1 h and 3 h.
Scale bar = 50 m.
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cultured on the polarized HAC was approximately 1.2 times
and 1.4 times larger than that on H-HAC 1 h and 3 h after
seeding, respectively. However, no significant differences in
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